Abstract: This research evaluates the molecular characterization and genotype structure analysis of endemic Hesperis isatidea populations using the Inter-Simple Sequence Repeat Polymerase Chain Reaction (ISSR-PCR) technique which were collected from various regions of Erzincan Province, Türkiye, and subsequently processed for genomic DNA isolation and ISSR-PCR analysis. Seven primers were used in the ISSR-PCR analyses and a total of 100 bands were obtained. Of these, 98 were polymorphic and two were monomorphic, and the polymorphism rate was determined to be 98%. The highest number of bands from the primers was obtained from UBC-855 and UBC-826, and the lowest number was obtained from UBC-836. The mean polymorphism information content (PIC) was determined to be 0.2886. The mean gene diversity value was determined to be 0.3559. The UPGMA dendrogram generated by PAUP 4.0b10 consists of two major clades. The genetic distance matrix values of samples from 5 different locations of Erzincan province, produced with the PAUP 4.0b10 program, were determined as 0.18000 and 0.470000, respectively. The PCA analysis created with the program PAST 4.06b consisted of four groups. The PCA analysis and the UPGMA dendrogram were compatible. These findings confirm that ISSR analysis is a highly polymorphic DNA-based marker system and an effective tool for differentiating between genotypes.